The present invention relates to methods for detecting and optionally quantitating multiple copies of a repeat sequence in a nucleic acid molecule, preferably a telomere or centromere repeat sequence. The invention also relates to a method for determining the replicative potential of a cell.
A large fraction of the DNA of all eukaryotes is made up of repeat sequences ranging from a few copies up to millions. Repeat functional sequences occur at the telomeres and centromeres of eukaryotic chromosomes. Telomeres and centromeres are important structural and functional elements of eukaryotic chromosomes. Telomeres are specialized nucleoprotein structures which are at the end of eukaryotic chromosomes (Blackburn, E H., Nature (London), 350:569-572, 1991). Telomeres in all vertebrates terminate in tandem arrays of the repeat sequence TTAGGG (Moyzis, R K., et al., Proc, Natl. Acad. Sci. USA 85:6622-6626, 1988). The repeat sequences are synthesized by the ribonucleoprotein enzyme telomerase, which is composed of both RNA and protein (Greider, C. W., and Blackburn, E. H. Cell 43:405-413, 1985; Morin, G B, Cell 59:521-529, 1989). In the absence of telomerase, telomeres shorten with cell divisions.
Telomeres have been shown to be critical for chromosome stability and function (Blackburn, E H., Nature (London), 350:569-572, 1991). Telomere loss has been shown to signal cell cycle arrest and chromosomal instability in yeast (Sandell, Ll, and Zakian, V A., Cell 57:633-643, 1989; and
Greider, C. W., and Blackburn, E. H. Cell 43:405-413, 1985). Telomeres in cells have been found to shorten with the age of the cell donor (Allsopp, R C. et al., Proc. Natl. Acad. Sci. USA 89, 10114-10118, 1992; Lindsey, J. et al., Mutat. Tes. 256:45-48, 1991, Vaziri, H., et al., Am. J. Hum. Genet. 52:661-667, 1993, Vaziri, H., et al., Proc. Natl. Acad. Sci. USA 91:9857-9861, 1994, and Hastie, N D., et al., Nature (London)., 346:866-868, 1990), Harley C B et al., Nature (London) 345:458-460, 1990; Harley, C B, Mutat. Res. 256:271-282, 1991), and this phenomenon has been implicated by some in aging (Harley, C B, Mutat. Res. 256:271-282, 1991), and in programmed cell death (Wright et al., Trends Genet 8:193-197, 1992). Abnormalities in telomeres have also been found in malignant cells (Meltzer et al., Nature Genetics 4:252-255, 1993).
The centromere region of mammalian chromosomes consists of tandem arrays of repetitive sequences which consist of various copy numbers of xcex1 satellite (Willard, H F, Trends Genet. 3:192-198, 1987), xcex2 satellite (Waye, J S., and Willard H F., Proc. Natl. Acad. Sci. USA 86:6250-6254, 1989) and the three classic satellites I, II and III (Prosser J. et al., J. Mol. Biol. 187:145-155, 1989).
Fluorescent in situ hybridization (FISH) techniques have been used to obtain information about the presence of telomeric and centromeric repeat sequences in chromosome preparations. Meyne and Moyzis (in Methods in Molecular Biology, Vol. 33: In Situ Hybridization Protocols, ed. K. H. A. Choo, 1994, Humana Press Inc., Totowa, N.J. USA) used FISH with synthetic oligonucleotide probes to confirm the presence of repeat telomere and centromere sequences. Directly labeled oligonucleotides should be attractive probes for FISH because of their small size (good penetration properties), single strand nature (no renaturation of probe required), and reproducible, controlled synthesis. However, these probes are not widely used for FISH because of their limited hybridization efficiency. The hybridization efficiency of oligonucleotide probes is modest because of their small size, and because conditions required for hybridization of the probe typically also favour the renaturation of denatured target DNA sequences with the longer homologous complimentary strands, resulting in direct and unfavourable competition for the oligo probe. Oligomer probes also have limited use as probes for single copy sequences.
Significantly, the studies using FISH have not provided optimal visualization of telomeres in a chromosome preparation nor have they provided information about the length of telomere or centromere regions. Southern analysis of the terminal restriction fragment (TRF) length distribution has so far been the only tool for studying telomere length (Allshire R C et al., Nucl. Acid Res. 17:4611-4627 and Harley C B et al., Nature 345:458-60). However, Southern analysis has a number of technical limitations. The TRF""s contain DNA other than the telomeric repeat sequences such as degenerate or non-TTAGGG as well as TTAGGG repeats in blocks other than at the distal end (Allshire et al., Nucl. Acid Res. 17:4611-4627; Counter et al., EMBO J. 11:1921-9, 1992 and Levy et al., J. Mol. Biol. 225:951-960, 1992). There is also interchromosomal variation in both non-TTAGGG and TTAGGG DNA in the TRF and variation in prior replicative histories in vitro or in vivo of cells in the population of cells required for TRF analysis, which makes it difficult to assess the relationship between cell senescence and TRF length in specific chromosome""s and in specific cell types. This is a particular problem in cancer where a variable or unknown number of normal cells may be present in a tumor sample.
It is apparent from the above discussion that there is a need for reliable methods for in situ visualization and quantification of telomere and centromere regions in chromosome preparations.
The present inventor has developed a method for detecting repeat sequences in the genome of a single cell or in an individual chromosome. This has been illustrated by the detection of telomeric repeat sequences at the end of human chromosomes. It was significantly found that the illustrated method results in the highly sensitive and efficient staining of all telomeres in a chromosome preparation. The present inventor also found a high correlation between fluorescence intensity of telomere sister chromatids in metaphase chromosomes. The method thus allows for the highly sensitive and efficient detection of telomeres in a chromosome preparation, and/or it allows for the quantification of the length of telomeric arrays at individual ends. The method also permits one skilled in the art to determine the effect of telomere loss on cell viability and chromosome behaviour in a variety of disease states.
The method of the invention can also be used to detect and/or quantitate the length of other repeat sequences for example, centromere repeats and polymorphisms of the telomere and centromere repeats.
The method of the invention addresses many of the technical limitations of conventional in situ hybridization procedures. The limitations of using oligonucleotide probes in in situ hybridization has been overcome by using nucleic acid analogue probes, and conditions that allow hybridization of the probes but prevent the renaturation of target DNA. The method of the invention provides a more efficient and sensitive procedure than existing or conventional in situ hybridization methods using DNA or RNA oligo probes.
Therefore, broadly stated the present invention relates to a method for detecting multiple copies of a repeat sequence in a nucleic acid molecule comprising (a) treating the nucleic acid molecule with a probe which is a nucleic acid analogue which is capable of hybridizing to the repeat sequence in the nucleic acid molecule and which is labelled with a detectable substance, under conditions permitting the probe to hybridize to repeat sequences in the nucleic acid molecule; and, (b) identifying probe hybridized to complementary repeat sequences in the nucleic acid molecule by directly or indirectly detecting the detectable substance.
In an embodiment of the invention, the detectable substance is a fluorophore, an image is formed of the probe hybridized to repeat sequences in the nucleic acid molecule, and, the multiple copies of the repeat sequences in the nucleic acid molecule are detected in the image.
The method of the invention may be used to quantitate the length of multiple copies of a repeat sequence in a nucleic acid molecule based on the direct relationship between the intensity of the signal produced directly or indirectly by the detectable substance, and the length of the multiple copies of the telomere sequence. Therefore, in accordance with a specific embodiment, the invention provides a method for quantitating the length of multiple copies of a repeat sequence in a nucleic acid molecule comprising (a) treating the nucleic acid molecule with a probe which is a nucleic acid analogue which is capable of hybridizing to the repeat sequence in the nucleic acid molecule and which is labelled with a detectable substance, under conditions permitting the probe to hybridize to repeat sequences in the nucleic acid molecule; (b) identifying probe hybridized to repeat sequences in the nucleic acid molecule by detecting a signal produced directly or indirectly by the detectable substance; and, (c) optionally, quantitating the length of the multiple copies of the repeat sequences in the nucleic acid molecule based on the direct relationship between the intensity of the signal produced directly or indirectly by the detectable substance and the length of the multiple copies of the repeat sequence.
The repeat sequence which is detected and/or quantitated is preferably a telomere or centromere repeat sequence, most preferably a human telomeric repeat sequence. In a preferred embodiment, multiple copies of the telomere repeat sequence TTAGGG in a nucleic acid molecule are detected using a probe containing the sequence CCCTAA.
In a particularly preferred embodiment of the invention, a method is provided for quantitating the length of multiple copies of the telomere repeat sequence TTAGGG in a nucleic acid molecule comprising (a) treating the nucleic acid molecule with a probe in the presence of a blocking reagent and a denaturing agent, preferably formamide, most preferably 70% formamide, and permitting the probe to hybridize to TTAGGG telomere repeat sequences in the nucleic acid molecule, wherein the probe is a nucleic acid analogue comprising the sequence CCCTAA and the probe is labelled with a fluorophore, and, (b) forming an image of probe hybridized to TTAGGG telomere repeat sequences in the nucleic acid molecule; and, (c) quantitating the length of the TTAGGG telomere repeat sequences in the nucleic acid molecule based on the direct relationship between fluorescence intensity and the length of the multiple copies of the telomere sequence.
The present invention also relates to a method for determining the replicative potential of a cell by quantitating the length of multiple copies of a telomere repeat sequence TTAGGG in nucleic acid molecules in the cell comprising (a) treating the nucleic acid molecules with a probe, under conditions permitting the probe to hybridize to telomere repeat sequences in the nucleic acid molecules, wherein the probe is a nucleic-acid analogue comprising the sequence CCCTAA and the probe is labelled with a detectable substance; (b) identifying probe hybridized to TTAGGG repeat sequences in the nucleic acid molecules by detecting signals produced directly or indirectly by the detectable substance; (c) quantitating the length of the multiple copies of the TTAGGG telomere repeat sequence based on the direct relationship between the intensity of the signals produced directly or indirectly by the detectable substance and the length of the multiple copies of the telomere repeat sequence and (d) determining the replicative potential by comparing the quantitated length of the multiple copies of the telomere repeat sequence with the length of multiple copies of the telomere repeat sequence associated with cells having a known replicative potential.
The invention still further contemplates a method for distinguishing normal cells from abnormal cells (e.g. tumor cells) in a cell suspension comprising: (a) treating the cells in the cell suspension with a probe which is a nucleic acid analogue which is capable of hybridizing to a repeat sequence in nucleic acid molecules in the nuclei of the cells and which is labelled with a detectable substance, under conditions permitting the probe to hybridize to repeat sequences in the nucleic acid molecules; (b) identifying probe hybridized to repeat sequences in the nucleic acid molecules by detecting a signal produced directly or indirectly by the detectable substance; and, (c) determining whether the cells are normal cells or abnormal cells by comparing the signal with a signal associated with known normal cells or abnormal cells. Preferably, the repeat sequence which is quantitated is a telomere repeat sequence, and the probe comprises the sequence CCCTAA.
In preferred embodiments of the methods of the invention, the detectable substance is a fluorophore, a digital image is created and the length of the multiple copies of the repeat sequence in the nucleic acid molecule is quantitated from the specific fluorescence intensity calculated from photon counts at defined positions within the digital image.
The methods of the invention may also be used to determine the effect of a substance on telomerase activity. Accordingly, the invention provides a method for determining the effect of a substance on telomerase activity comprising (a) treating cells having telomerase activity with a probe which is a nucleic acid analogue comprising the sequence CCCTAA and which is labelled with a detectable substance, preferably a fluorophore, and with a substance suspected of affecting telomerase activity, under conditions permitting the probe to hybridize to TTAGGG telomere repeat sequences in nucleic acid molecules in the cells; (b) identifying probe hybridized to TTAGGG telomere repeat sequences in the nucleic acid molecule by directly or indirectly detecting the detectable substance; (c) quantitating the length of multiple copies of the TTAGGG telomere repeat sequence in the nucleic acid molecules based on the direct relationship between the intensity of the signal produced directly or indirectly by the detectable substance and the length of the multiple copies of the telomere repeat sequence, and (d) determining the effect of the substance by comparing the quantitated length of the multiple copies of the telomere repeat sequence with the length of multiple copies of the telomere repeat sequence quantitated for the preparation in the absence of the substance.
The method of the present invention may be adapted to detect and/or quantitate multiple copies of repeat sequences in nucleic acid molecules in individual cells in suspension. In particular, the invention provides a method for quantitating multiple copies of repeat sequences in nucleic acid molecules in cells in suspension comprising; (a) treating the cells with a probe which is a nucleic acid analogue which is capable of hybridizing to the repeat sequences in the nucleic acid molecules and which is labelled with a detectable substance, under conditions permitting the probe to hybridize to repeat sequences in the nucleic acid molecules; (b) subjecting the treated cells to flow cytometry or image cytometry to detect the detectable substance and produce a signal corresponding to the amount of probe hybridized to repeat sequences in the nucleic acid molecules; and (c) optionally, quantitating the length of the multiple copies of the repeat sequences in the nucleic acid molecules based on the relationship between the intensity of the signal and the length of the multiple copies of the repeat sequences. In an embodiment, the method is used to quantitate multiple copies of a telomere repeat sequence TTAGGG. Preferably, the detectable substance is detected using flow cytometry which has the advantage that thousands of nuclei can be measured per second, and multiple other cell parameters (such as DNA content) can be analyzed simultaneously. The method is particularly suited for analyzing normal and abnormal cells, including lymphocytes, from blood and bone marrow.
The invention also contemplates a method for distinguishing normal cells from abnormal cells (e.g. tumor cells) in a suspension containing normal cells and abnormal cells comprising: (a) treating the cells in the cell suspension with a probe which is a nucleic acid analogue which is capable of hybridizing to a repeat sequence in nucleic acid molecules in the nuclei of the cells and which is labelled with a detectable substance, under conditions permitting the probe to hybridize to repeat sequences in the nucleic acid molecules; (b) subjecting the treated cells to flow cytometry or image cytometry to detect the detectable substance and produce a signal corresponding to the amount of probe hybridized to repeat sequences in the nucleic acid molecules; and (c) determining whether the cells are normal cells or abnormal cells by comparing the signal with a signal obtained for known normal cells or abnormal cells.
The invention also contemplates novel Peptide Nucleic Acid (PNA) probes. In particular, the invention relates to a Peptide Nucleic Acid (PNA) probe comprising the following sequence: TTAGGG, CCCTAA, CCCCAA, CCCCAAAA, CCCACA, CCCTAAA, CCCCT, or CCATT. Preferred Peptide Nucleic Acid (PNA) probes of the invention include CCCTAACCCTAA [SEQ ID no: 1], CCCTAACCCTAAACCTAA [SEQ ID no: 2], TTAGGGTTAGGG [SEQ ID no: 3], TTAGGGTTAGGGTTAGGG [SEQ ID no: 4], CCATTCCATTCCATTCATT [SEQ ID no: 5], CCCATAACTAAACA [SEQ ID no: 6], GAGAATTGAACCACCG [SEQ ID no: 7], TTCCCTGCCGTTCG [SEQ ID no: 8], (GGC)5 [SEQ ID no: 13], or (TCG)5 [SEQ ID no: 14].
The invention also contemplates kits for performing the methods of the invention.
These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings. In addition, reference is made herein to various publications, which are hereby incorporated by reference in their entirety.